RNA regulation allows rapidly controlling gene expression in response to stimuli, including environmental changes. This project seeks to generate and utilize structural information to enhance our understanding of these processes with an emphasis on the importance of RNA target specificity for proper gene regulation. In this fiscal year, we have studied the atomic structures and functions of classical PUF proteins, which are sequence-specific RNA-binding proteins that are important regulators of gene expression for embryonic development and germline stem cell maintenance, and discovered new PUF proteins that function to regulate pre-ribosomal RNA processing in ribosomal biogenesis. Beginning with determining the first crystal structure of a PUF protein in complex with RNA to recent work on the specificity of human, yeast, and C. elegans proteins, we have identified both common and unique features of RNA recognition by this family of proteins. The combination of the features in any particular protein results in a unique network of mRNAs that are regulated by that protein. We have advanced this work by identifying and studying the crystal structures and RNA target specificity of additional PUF protein family members. We have identified the broadened RNA sequence specificity of yeast Puf5 protein. This work is described in a manuscript in press at Nature Communications. We have also used crystal structures, biochemical assays, and in vivo studies to identify new families of PUF-related proteins that operate in ribosomal biogenesis. We have discovered that human Puf-A protein and yeast ortholog Puf6 protein function in ribosomal biogenesis. These new PUF-like proteins have different RNA recognition properties than the classical PUF proteins. They bind to either single-stranded or structured RNAs and lack sequence specificity.